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Genetic and environmental factors affecting the incidence of coronary artery disease in heterozygous… Hill, John Stuart 1990

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G E N E T I C AND ENVIRONMENTAL FACTORS A F F E C T I N G T H E INCIDENCE OF CORONARY A R T E R Y DISEASE IN H E T E R O Z Y G O U S FAMILIAL H Y P E R C H O L E S T E R O L E M I A by J O H N STUART H I L L B.M.L.Sc, University of British Columbia, 1988 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard T H E UNIVERSITY O F BRITISH C O L U M B I A August 1990 ®John Stuart Hill, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology The University of British Columbia Vancouver, Canada Date August 1990.  DE-6 (2/88) ABSTRACT Fami l i a l hypercholesterolemia ( F H ) is an autosomal dominant disorder in which the primary defect is a mutation in the L D L receptor. Heterozygous F H is among the most common inborn errors of metabolism and remains as the best example of an inherited defect causing premature coronary artery disease ( C A D ) . This thesis describes the physical and biochemical characteristics of heterozygous F H i n a large cohort consisting of 208 women and 156 men. The influence of both genetic and environmental factors on the clinical expression of F H were investigated to better understand the phenotypic variation within F H and thus improve the prediction and treatment of C A D in affected individuals. The general incidence of C A D in this population was lower compared to previous reports but the differences between the sexes were expected. It was shown that men had a much higher frequency of C A D (31%) compared to women (13%) despite having lower concentrations of total and L D L cholesterol. In addition, the average age of onset of coronary symptoms was delayed in females, 55 years compared to 48 years for males. A greater risk of developing C A D for men was associated with low levels of H D L cholesterol and a history of smoking. In women, however, C A D was associated with elevated triglyceride levels and the presence of hypertension. In order to efficiently assess the influence of the co-inheritance of the apolipoprotein E polymorphism in this large F H population, a novel apo E phenotyping procedure was developed. Phenotypes were determined directly from plasma which was neuraminidase treated, delipidated and focused in polyacrylamide i i minigels. The accuracy of this method was confirmed by making a comparison to the established procedure of phenotyping by isoelectric focusing of delipidated V L D L . The low cost, speed and simplicity of the minigel methodology provided ideal conditions to phenotype a large patient population. The frequencies of the el, e3 and e4 alleles of apolipoprotein E in 125 unrelated F H subjects did not differ significantly from the normal population. In addition, there was no apparent relationship between apo E4 and the concentration of any of the parameters in the plasma lipid profile. However, the presence of the E2 isoform was associated with significantly elevated triglycerides in both sexes. From this study, it is evident that the mutant F H gene exerts its effect within a system of interacting environmental and polygenic factors that are known to modify atherosclerotic risk. It has been established that the dissimilarity in the frequency of C A D between men and women is related to differences between the impact of known risk factors and the incidence of C A D . Therefore, the importance of the influence of these risk factors and the differences between men and women should be emphasized when treating and predicting the development of C A D in patients with F H . iii T A B L E O F C O N T E N T S A B S T R A C T ii T A B L E O F C O N T E N T S iv A B B R E V I A T I O N S vii LIST O F T A B L E S viii LIST O F F I G U R E S ix A C K N O W L E D G E M E N T S x 1 INTRODUCTION 1 1.1 F A M I L I A L H Y P E R C H O L E S T E R O L E M I A 1 1.1.1 Historical perspectives 1 1.1.2 Homozygous and heterozygous F H 2 1.1.3 Biochemistry of the L D L receptor 2 1.1.4 Genetics of the L D L receptor 4 1.1.5 Diagnosis of heterozygous F H 6 1.1.6 Phenotypic variation in F H 7 1.1.6.1 Lipid profile 7 1.1.6.2 Xanthomas 8 1.1.6.3 Coronary artery disease (CAD) 8 1.2 B I O C H E M I C A L G E N E T I C S O F A P O L I P O P R O T E I N E 9 1.2.1 Methods of apo E phenotyping 10 1.2.2 Allelic effect in the normal population 12 1.2.3 Apolipoprotein E and hyperlipidemia 12 1.3 R A T I O N A L E 14 iv 1.4 S P E C I F I C A I M S 16 2 MATERIALS AND METHODS 17 2.1 M A T E R I A L S 17 2.2 Subjects Studied 17 2.3 Plasma preparation and l ip id analyses 18 2.4 A p o E phenotyping of delipidated V L D L 18 2.4.1 Sample preparation 19 2.4.2 Isoelectric focusing 19 2.4.3 G e l staining 20 2.5 A p o E Phenotyping directly from plasma 20 2.5.1 Sample preparation 20 2.5.2 Isoelectric focusing 21 2.5.3 Immunoblotting 21 2.6 D a t a Analysis 22 3 RESULTS 24 3.1 A p o E Phenotyping 24 3.1.1 Neuraminidase treatment 24 3.1.2 Comparison of traditional and new phenotyping methodologies . 25 3.1.3 A l l e l e frequencies in a population from Vancouver 26 3.1.4 A p o E phenotype and l ip id profile 27 3.2 F H Populat ion 30 3.2.1 A g e and sex distribution 30 3.2.2 Plasma lipids, lipoproteins and apoproteins 31 3.2.3 Cl in ica l Features of F H 34 v 3.2.4 Plasma lipids, lipoproteins and apoproteins for those with and without C A D 37 3.2.5 Frequency of risk factors for the C A D ( + ) and C A D (-) groups 38 3.2.6 Apolipoprotein E and F H 40 4 DISCUSSION 43 4.1 Apo E Phenotyping Methodology 43 4.2 F H Population 45 5 R E F E R E N C E S 52 vi ABBREVIATIONS ACAT acyl CoA: cholesterol acyltransferase apo apolipoprotein BMI body mass index CAD coronary artery disease CoA coenzyme A DNA deoxyribonucleic acid FH familial hypercholesterolemia HDL high density lipoprotein HDL-C high density lipoprotein cholesterol HMG-CoA 3-hydroxy-3-methylglutaryl CoA IEF isoelectric focusing LDL low density lipoprotein LDL-C low density lipoprotein cholesterol MI myocardial infarction RFLPs restriction fragment length polymorphisms TBS tris-buffered saline TC total cholesterol TG triglyceride VLDL very low density lipoprotein vii LIST OF TABLES Table 1. Apolipoprotein E phenotype assignments using two different methodologies 26 Table 2. Apolipoprotein E phenotype and allele frequencies in a normal population from Vancouver 27 Table 3. Apolipoprotein E phenotype and levels of lipids, lipoproteins and apoproteins 28 Table 4. Estimation of the average effects of the three apo E alleles on the levels of lipids, lipoproteins and apoproteins, expressed as a percentage of the respective total population means 29 Table 5. Lipids, lipoproteins and apoproteins in F H patients 32 Table 6. Mean values for lipids, lipoproteins and apoproteins for each age division in F H 33 Table 7. Clinical data for each age division in F H 35 Table 8. Lipids, lipoproteins and apoproteins in F H patients with and without C A D 38 Table 9. Assessment of the effects of smoking, hypertension, H D L cholesterol levels and BMI on CAD in F H . 39 Table 10. Apo E phenotype distribution and allele frequency in F H 40 Table 11. Clinical data in F H patients with and without apolipoprotein E4 and E2 41 Table 12. Lipid, lipoprotein and apoprotein levels in F H patients with and without apolipoprotein E4 and E2 42 viii LIST OF FIGURES Figure 1. Cellular path of the L D L receptor 3 Figure 2. Mutations in the L D L receptor gene 5 Figure 3. Four classes of mutations at the L D L receptor locus 6 Figure 4. Apo E immunoblots depicting the six common phenotypes 24 Figure 5. Age distribution of F H patients for each sex 30 Figure 6. The cumulative frequency of CAD in males and females with F H 36 Figure 7. The distribution of the age of onset of symptoms of CAD in F H 37 ix A C K N O W L E D G E M E N T S I would l ike to express my appreciation to everyone who has contributed to the successful complet ion of these studies. Specifically, I wish to thank my supervisor, D r . Haydn Pritchard, who has consistently provided invaluable support and guidance throughout my academic studies and scientific research. I would also l ike to express my gratitude to the members of my supervisory committee, in particular, D r . Michae l Hayden and D r . J i r i F roh l ich for their recommendations regarding the methods of design and analysis of the F H population study. In addition, I would l ike to thank Cathy McGuinness and Roger M c L e o d for their helpful discussions and technical assistance during the development of the new apolipoprotein E phenotyping methodology. A l s o , I wish to express a special thanks to my parents for offering support and encouragement throughout my studies. Final ly, I would l ike to express my gratitude to the Bri t ish Co lumbia and Y u k o n Hear t foundation for financial support during this period. x 1 INTRODUCTION 1.1 FAMILIAL H Y P E R C H O L E S T E R O L E M I A Fami l i a l hypercholesterolemia ( F H ) is a congenital disorder i n which the primary defect is a mutation in the gene which encodes for the receptor of plasma low density l ipoprotein ( L D L ) . A deficiency or absence of these cell surface receptors causes the levels of plasma L D L cholesterol to rise leading to an increase in the deposition of cholesterol i n tissues. Physical signs of this cholesterol accumulation include arcus senilis and tendon xanthomas as wel l as atheromas which contribute to the premature development of atherosclerosis. This genetic defect is transmitted as an autosomal dominant gene (1) and remains as the best illustration of an inherited disorder causing coronary heart disease. 1.1.1 Historical perspectives The clinical characteristics of this disease and its familial nature were first described i n the 1930s by M u l l e r (2) and Thannhauser et al . (3). M o r e extensive family studies by Aldersberg (4), Hirschorn and Wi lk inson (5) and others (6,7) in the 1940s and 1950s confirmed that a genetic basis for hypercholesterolemia existed. Distinguishing the heterozygotes from homozygotes was clearly established by Khachadurian (8) in the 1960s and thus provided evidence for the single gene inheritance of this disorder. In the 1970s, technical developments related to the use of cultured fibroblasts enabled Brown and Goldste in (9) to identify the basic biochemical defect of a decreased or absent receptor activity for L D L particles. Advances i n molecular biology i n the early 1980s allowed identification of the L D L receptor gene (10) and eventually the definition of specific genetic defects which 1 made possible the determination of the structural basis of the various mutations. 1.1.2 Homozygous and heterozygous F H The frequency of heterozygous F H is estimated to be 1 in 500 persons, placing F H among the most common in born errors of metabolism (11). Heterozygotes can be characterized clinically from birth by elevated levels of plasma cholesterol and often develop tendon xanthomas and corneal arcus after age 20 (12) while symptoms of coronary heart disease appear in the fourth decade. In contrast, the clinical picture of homozygotes is remarkably consistent and distinctly different compared to heterozygotes. Homozygous F H occurs in about one in one million persons and manifests itself by the presence of severe hypercholesterolemia at birth (16-26 mmol/L) and the appearance of distinct yellow-orange cutaneous xanthomas which develop in all subjects by age 4 (13,14). These individuals have a poor prognosis and often die from coronary events before the age of 30 (13,14). 1.1.3 Biochemistry of the L D L receptor Figure 1 illustrates the cellular path of the L D L receptors which originates with its synthesis in the endoplasmic reticulum. This precursor protein then travels to the Golgi complex for further processing. After transport to the plasma membrane, the receptors cluster in coated pits on the surface of the cells. The receptor binds L D L by interacting with apo B-100 (15) and is taken up through a receptor mediated endocytosis. After formation of the endosome, the receptors can dissociate from L D L to form a recycling vesicle which returns to the cell surface. Subsequently, the L D L is degraded in lysosomes permitting the release of cholesterol and amino acids for further cellular metabolism. It is cholesterol derived from this hydrolysis reaction 2 which modulates a sophisticated system of feedback control that regulates the intracellular cholesterol concentration (16). For example, the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), the rate controlling enzyme of cholesterol biosynthesis, is suppressed and thus cholesterol synthesis within the cell is inhibited (17). In addition, the enzyme called acyl CoA: cholesterol acyltransferase (ACAT) is activated in order that excess cholesterol may be stored as cholesterol esters. The third effect is to terminate the synthesis of L D L receptors and thereby protect the cells from accumulating too much cholesterol (18). Figure 1. Cellular path of the LDL receptor. The receptor is synthesized in the endoplasmic reticulum from which it travels to the Golgi complex, cell surface, coated pit, endosome and back to the surface (19). 3 A deficiency of L D L receptors causes a decrease in the rate of removal of L D L particles from plasma and, as a result, the level of L D L rises in inverse proportion to the number of functional receptors. F H heterozygotes possess one normal allele and one mutant allele and thus their cells are able to bind and internalize L D L at approximately half the normal rate. In contrast, homozygotes have two mutant alleles at the L D L receptor locus and thus their cells demonstrate a total or near total inability to bind or take up L D L . Since there are many different mutations affecting the function of the L D L receptor gene in the general population (20), many phenotypic homozygotes are in fact compound heterozygotes for two different mutations. However, in all cases, there is an excess pool of circulating plasma L D L which is deposited in connective tissue and scavenger cells, producing xanthomas atheromas and ultimately coronary disease. 1.1.4 Genetics of the L D L receptor The L D L receptor gene is located on the short arm of chromosome 19 and is made up of 18 exons spanning 45 kB (21,22). The gene product is a single-chain glycoprotein containing 839 amino acids in its final mature form (23). In most cases the exon sequences of the gene correspond to the structural domains of the protein. As shown in Figure 2, 16 different mutations in the L D L receptor gene have been identified. They include large insertions and deletions as well as single base differences that create missense or nonsense codons. Although the mutations are distributed over the entire 45 kB of the gene and affect all of the structural domains of the protein, they can still be characterized as falling into one of four broad classes. 4 6 kb 4 k b 5.5kb French Canadian > 10 kb Exon No. Signal Sequence 3 bp 14kb 12bp .8kb I i l l iff 3 4 5 6 Ligand Binding T I n s e r t i o n D e l e t i o n e N o n s e n s e ' a M i s s e n s e 5kb 7.8 kb 4kb Lebanese Mil / / / / V / / / B 4nt 7 8 9 10 11 12 1314 15 I EGF Precursor Homology 16 17 18 L C-L inked Sugars Cytoplasmic Membrane • Spanning Figure 2. Muta t ions in the L D L receptor gene. A schematic of the normal gene is shown in the center outlining the exons (hatched boxes) and introns (connecting lines) as well as the corresponding structural domains of the mature protein. The different mutations are indicated above the diagram with a description of the type of symbol found in the key in the box (24). These different categories of mutations of the L D L receptor have been identified based on the phenotypic behaviour of the mutant proteins (Figure 3). The most common of these mutations are class 1 alleles which fail to produce any immunoprecipitable protein (null alleles). Class 2 alleles produce proteins which are blocked in intracellular transport between the endoplasmic reticulum and the Golgi complex. Class 3 alleles encode for proteins which are synthesized and transported to the cell surface but fail to bind L D L normally. Class 4 alleles, the rarest, encode for 5 proteins which reach the cell surface and bind L D L normally but fail to cluster in coated pits and therefore do not internalize bound L D L . ENDOPLASMIC RETICULUM Class of Mutation Synthesis Transport from ER to Golgi Binding of LDL Clustering in Coated Pits ® X ® - • X CD ^ - Y ^ A ® 1 Figure 3. Four classes of mutations at the L D L receptor locus. The mutations affect synthesis transport, binding and clustering of the L D L receptor (25). 1.1.5 Diagnosis of heterozygous F H A s there are several causes of high L D L cholesterol levels, its presence does not distinguish those patients with F H . To confirm this diagnosis, hypercholesterolemia in association with tendon xanthomas or a family history of hypercholesterolemia associated with tendon xanthomas should be evident. A s a result, it is difficult to identify those heterozygotes lacking tendon xanthomas in the absence of a complete family analysis. Alternatively, the diagnosis can be based on the demonstration of a 6 the demonstration of a deficiency in receptor activity or a mutation in the L D L receptor gene. Within a family, a mutation can be traced by D N A variants or restriction fragment length polymorphisms (RFLPs) that are closely linked to the gene (26). Such D N A markers could be used in family studies to detect F H heterozygotes in individuals with border line elevations in plasma cholesterol. However, such an approach is not feasible in the general population as many different F H mutations have been described and it is likely that many more still exist. Unless these methods of analysis improve, the most practical approach to forming a diagnosis is based on an individual's clinical features in the presence of a proven family history. 1.1.6 Phenotypic variation in F H In homozygotes, phenotypic expression of the disorder is dominated by genotypic variation at the L D L receptor gene locus with other influences exerting relatively little effect. In contrast, phenotypic variation in heterozygotes is influenced not only by the nature of the underlying gene mutation but also, by gender, environmental factors and possibly other forms of genetic polymorphism. 1.1.6.1 Lipid profile Although there is considerable variation in plasma cholesterol levels among individual F H heterozygotes, there is a remarkable consistency in the mean population values in different parts of the world. For example, 8.5 mmol/L in Lebanon (13), 9.1 mmol/L in Japan (27), 8.9 mmol/L in Canada (28) and 9.5 mmol/L in the United States (12). Mean H D L cholesterol levels in F H patients tend to be lower than the normal population (12,28-30) but an explanation for this 7 reduction has not been established. Several studies suggest that although some heterozygotes have a moderate elevation in triglycerides, the mean value is not significantly greater than the general population (12,14,31). In contrast, however, studies of F H in French Canadians (28,29) have indicated that hypertriglyceridemia was a common feature in this population. None of these studies have indicated any significant difference in the levels of total or L D L cholesterol between the sexes. However, it has been shown in one report that males with F H have lower plasma concentrations of H D L cholesterol compared to females (28) and in the same study higher triglyceride levels were observed in men with F H . 1.1.6.2 Xanthomas Tendon xanthomas are the hallmark sign of this disease as they are found almost exclusively in F H . The most common site are the Achilles tendons but they are also often seen in the extensor tendons of the hands. The frequency of tendon xanthomas increases as a function of age and has been found to be equal in both sexes (28,32). It is evident that the rate of deposition of the LDL-derived cholesterol is proportional to the severity and duration of the elevation in L D L , but additional unknown factors are also thought to have a role (8,14). 1.1.6.3 Coronary artery disease (CAD) In several different populations, the greater incidence and premature onset of C A D in F H has been well described (28,32-35). In addition, it has been shown that females have a much lower incidence of C A D (28,33,34) and a later onset of symptoms compared to males (28,32,35,36). From an analysis of 276 affected 8 patients, Stone et al. found the cumulative probability of C A D in males was 16% at age 40 and 52% at age 60; however, in females, the cumulative risk was 33% by age 60 (37). The increased frequency and severity of C A D in F H males parallels those observations seen in the general population and thus, the F H population may be affected by the same risk factors. Although all patients who are heterozygous for F H present with high cholesterol, the severity of the disease in terms of coronary symptoms is quite varied and not necessarily correlated with serum cholesterol levels. In fact, lower concentrations of H D L cholesterol in F H patients of both sexes have been reported to have greater predictive value for the presence of C A D than levels of total or L D L cholesterol. Also, an elevation in triglycerides in females has been correlated with C A D (34). In addition, very few studies have addressed risk factors such as smoking, obesity, and hypertension and their role in the development of C A D . However, a single study revealed that smoking in males and hypertension in females were related to C A D and suggested that their presence could explain the observed differences in the incidence of disease between the sexes (32). Also, it is likely that genetic influences separate from those of the L D L receptor gene could contribute to the phenotypic diversity of F H . In particular, the well described polymorphism of apolipoprotein E, structure and function, may be one of the factors which determines the heterogeneity of the clinical expression in this disorder. 1.2 B I O C H E M I C A L GENETICS O F APOLIPOPROTEIN E Apolipoprotein E is a structural component of plasma chylomicrons, very low density lipoprotein ( V L D L ) and high density lipoprotein (HDL) (38). It has a major 9 regulatory role in the lipid metabolism of these lipoprotein particles via specific apo E receptors and by L D L (apo B,E) receptors on the liver and other peripheral tissues (39). As a ligand for these receptors, apo E is responsible, in part, for the uptake of dietary cholesterol in the form of chylomicron remnants, for the clearance of V L D L remnants and for the removal of excess cholesterol from peripheral tissues through the hepatic clearance of H D L containing apo E . It is synthesized primarily in the liver, but it is also produced by a number of different tissues in the body (40). The newly synthesized preprotein undergoes intracellular proteolysis, glycosylation and desialylation resulting in a single polypeptide chain of 299 amino acids with a calculated molecular weight of 34,145 (41,42). The gene locus for plasma apolipoprotein E , found on chromosome 19 (43), is polymorphic having three common alleles (e2,e3,e4) which encode the three major isoforms of apo E found in plasma: apo E2, apo E3 and apo E4 (44). The three major isoforms differ by amino acid substitutions at one or both of two sites (residues 112 and 158) on the 299 amino acid chain (45). E4 differs from E3 by the replacement of cysteine with arginine at position 112. E2 differs from E3 by the replacement of arginine with cysteine at position 158. As a result, apo E4 has one more positive charge compared to apo E3, while apo E2 has one less. The three apo E alleles code for three protein isoforms which result in the expression of three homozygous and three heterozygous phenotypes: E4/4, E3/3, E2/2, E4/3, E4/2 and E3/2. 1.2.1 Methods of apo E phenotyping The single charge differences in the common apo E isoforms allow their 10 separation by isoelectric focusing (IEF). Traditionally, apo E phenotype has been determined by IEF of ultracentrifugally isolated V L D L (46). However, this method is labour-intensive and requires a large amount of plasma. In addition, the need for ultracentrifugation adds to the cost of this method and prevents its application to studies of larger populations. As a result, more efficient and practical procedures for phenotyping are required. More recently, there have been approaches to eliminate the need for ultracentrifugation by combining immunoblotting with serum delipidation (47,48), serum treated with guanidine-HCl (48), charge-shift electrophoresis of hydrophobic serum proteins (49), and dialysed serum (50). A second advantage of these procedures is the ability to use smaller volumes of plasma or serum since the IEF band pattern is visualized by an anti-apo E antibody. However, in all cases there are instances where the final band patterns achieved remain difficult to interpret due to the presence of additional sialylated isoforms. In order to resolve this ambiguity, three different methods can be applied. First, one could perform a two dimensional electrophoresis of delipidated V L D L where the sialylated forms have a slightly higher molecular weight and thus can be differentiated from the asialo forms (51). Second, the sample can be treated with cysteamine which adds a positive charge to the sulfhydryl group of cysteine residues. This method will help to confirm the nature of the isoforms in both the homozygous and heterozygous states through a comparison of band intensities with the original isoelectric focusing pattern (45). The third method uses neuraminidase to remove the sialic acid residues before focusing (51). A new approach has utilized the polymerase chain reaction and allele-specific 11 nucleotide probes (52-54) or restriction isotyping (55) for apolipoprotein E genotyping. However, these methods require an initial DNA extraction step and more expensive equipment and reagents and therefore have not yet been established as viable clinical tests. Regardless of the chosen procedure, when attempting to phenotype a large population it is important that the method should be able to be performed in a relatively short time span and produce reliable results at low cost. 1.2.2 Allelic effect in the normal population In normal individuals, several studies have established that, compared to E3, E2 is associated with lower plasma cholesterol levels whereas individuals with apo E4 have higher cholesterol levels (56,57). These observations have been related to functional differences between the three isoforms. It has been shown that apo E2 has a significantly decreased ability to bind to either LDL or apo E receptors (45) resulting in a reduced in vivo catabolism of apo E2 (58). In contrast, apo E4 demonstrated an increased in vivo catabolism (59), even though it is indistinguishable from apo E3 in in vitro receptor binding activity (45). The relationship between apo E phenotype and mean cholesterol levels has been shown to be consistent among several studies from different parts of the world. These reports suggest that even differences in environmental factors and genetic background within these populations have little effect on the influence of apo E polymorphism in determining individual differences in plasma cholesterol. There have been no other genes identified which have such a large contribution to the determination of plasma cholesterol levels in the normal population. 1.2.3 Apolipoprotein E and hyperlipidemia 12 Studies have revealed that different apo E phenotypes are associated with specific lipoprotein profiles. For example, the majority of subjects with dysbetalipoproteinemia (6 migrating V L D L ) are apo E2 homozygotes (60), although it is estimated that less than 5% of E2/2 individuals have type III disease (61). This disorder is characterized by palmar and tuboeruptive xanthomas, premature peripheral vascular and coronary artery disease and accumulation of abnormal cholesterol-rich V L D L and IDL (62). The molecular basis of this disorder is thought to involve the decreased ability of apo E2 to bind to lipoprotein receptors (63,64) in conjunction with other genetic or environmental factors. Type III disease has also been associated with several different mutations of apo E (64-70) while other genetic variants found in the apo E gene have been identified with different forms of hyperlipoproteinemia (71-74). However, the relationship between the apo E alleles and atherosclerosis is still controversial. There have been conflicting reports on the association of the e4 allele with myocardial infarction (75-78) although a recent study has shown that the incidence of both the e2 and e4 alleles was greater in patients with ischemic heart disease (79). Previous reports have attempted to study the effects of the co-inheritance of these different alleles on the phenotypic expression of F H , but no clear pattern has been established (80-83). However, the visible relationship of the apo E gene with lipid levels and hyperlipidemia suggests that this polymorphism could alter the genetic risk of developing atherosclerotic disease. 13 1.3 R A T I O N A L E The current clinical and biochemical features of heterozygous familial hypercholesterolemia have been based on a variety of population studies. However, the relatively small population size in many of these reports may not always be sufficient to provide an accurate assessment of this disease. In addition, since the stringency of selection criteria differs among several studies, it is likely that some degree of heterogeneity exists in these populations with respect to the genetic cause of hypercholesterolemia. In contrast, the studies of single kindreds with F H provide distinct advantages; however, such an analysis reflects the function of a single mutation only. Consequently, the characteristics of this defect may not necessarily be a good representation of the general features of the disease. Therefore, the qualities of the selected population must be kept in perspective when analysing these data as the extrapolation of such results in definitively describing the characteristics of F H may not be appropriate. It is the aim of this study to carry out a detailed examination of the physical and biochemical features of a large cohort of individuals with heterozygous F H . The application of specific selection criteria should ensure that no other causes of hypercholesterolemia secondary to F H are included. In addition, the diversity in ethnic origin and size of this population would indicate that it is likely to consist of a large variety of mutations in the L D L receptor gene. As a result, the design of this investigation should produce results which are representative of F H in the general population. In order to better understand the differences in the clinical expression of F H , it 14 is necessary to determine the role of the common risk factors associated with C A D . This is achieved through a detailed description of the influence of gender on the concentration of lipids, in the sensitivity to smoking, obesity and hypertension and ultimately on the incidence of coronary artery disease. It is well established that allelic variation at the apo E locus affects cholesterol metabolism in both normal and dyslipidemic individuals. However, the impact of the co-inheritance of these different alleles with F H remains unresolved. In order to better assess and predict the clinical expression of F H , it is important to establish the magnitude of the influence of the apo E polymorphism. In this study, it is proposed that the frequency of the three different alleles in individuals with F H will not differ when compared to a normal population. However, it is hypothesized that the presence of the E 4 isoform could adversely affect the clinical expression of F H . The nature of this unique study enables the identification of those factors which in addition to total cholesterol levels are associated with the development of premature coronary atherosclerosis in heterozygous F H . 15 1.4 SPECIFIC AIMS 1. To define F H and identify those patients from the Lipid Clinic population who satisfy the selection criteria. 2. To describe the biochemical and physical presentation of F H in a large heterogenous population. 3. To determine the effects of gender, lipid profile and known risk factors on the incidence of coronary disease in F H . 4. To establish a novel apolipoprotein E phenotyping methodology which can be performed for a large number of samples. 5. To determine the frequency distribution of the apo E alleles in both the normal control and F H populations. 6. To assess the effects of the apolipoprotein E polymorphism on the biochemical and physical presentation of F H . 16 2 MATERIALS AND M E T H O D S 2.1 MATERIALS Clostridium perfringens type V neuraminidase, 3,3-diaminobenzidine hydrochloride(DAB), cobalt chloride(CoCl2), and Nonidet P-40 were purchased from Sigma Chemical Co., St. Louis M O . Ampholytes (pH 5.0-6.5, p H 4.0-6.0) were obtained from Pharmacia L K B Biotechnology Uppsala, Sweden. Electrophoresis grade reagents including urea, acrylamide, N,N'-methylenebisacrylamide, dithiothreitol (DTT), ammonium persulfate and T E M E D were from BioRad Laboratories, Richmond, California. Nitrocellulose paper was from Schleicher and Schuell, Keene,N.H. Goat-anti-human apo E antiserum was purchased from Daiichi Chemicals, Tokyo, Japan. Protein G (GammaBind G) conjugated to horseradish peroxidase was obtained from Genex corp., Gaithersburg,MD. Al l other chemicals were of analytical grade, from B D H Chemicals Canada Ltd., Vancouver, B.C. 2.2 Subjects Studied In order to evaluate a novel apo E phenotyping procedure, 51 plasma samples were obtained from patients attending the University Hospital Lipid Clinic which were then divided and analysed by the current routine procedure (focusing of delipidated V L D L ) (46) and the new methodology. In addition, specimens from a randomly selected healthy normal population of 203 subjects aged 18-78 years living in the Vancouver area were analysed for lipids and apo E phenotype. A total of 364 patients from 283 families with heterozygous familial hypercholesterolemia were identified among a population in the University Hospital Lipid Clinic. F H was diagnosed if subjects satisfied the following criteria: 17 1) A level of LDL-cholesterol > 95th percentile corrected for both age and sex 2) tendon xanthomas in the patient or a first degree relative. The criteria for C A D were the presence of angina or myocardial infarction (MI) or angiographically proven disease or a history of coronary bypass surgery. Diagnosis of angina and MI were made on the basis of documented medical records. Smoking was defined in both former and current smokers who had a history of smoking > 5 pack years where 1 pack year is equivalent to smoking 1 pack/day for 1 year. Hypertension was indicated if clinically documented even if patients were currently on anti-hypertensive medication. The body mass index (BMI) was calculated as body weight in kilograms divided by the square of the height in meters. Plasma samples from 125 unrelated F H subjects from the larger patient population were randomly chosen for apo E phenotype determination. 2.3 Plasma preparation and lipid analyses Venous blood was collected from all subjects after an overnight fast of 12-16 hr. The E D T A plasma was separated from cells by low speed centrifugation (1,200 x g, 20 minutes) and frozen at -70°C before analysis. Total cholesterol (TC) and triglycerides (TG) were measured by established enzymatic techniques (84,85). H D L -cholesterol (HDL-C) was determined as cholesterol remaining after precipitation of apo-B containing lipoproteins with heparin-MnCl 2 (86). L D L - C was calculated from the formula: (TC - H D L - C ) - TG/2.2 where all values were measured in mmol/L. Plasma apo A-I and apo B were measured by rate nephelometry using a Beckman Immunochemistry System (87). 2.4 Apo E phenotyping of delipidated V L D L 18 2.4.1 Sample preparation Six ml of plasma was subjected to ultracentrifugation at a density of 1.006 g/ml at 105,000g for 18 hr. at 15°C. V L D L was separated by tube slicing and then washed at a density of 1.006 g/ml at 105,000g for 18 hr. at 15°C. After tube slicing, the V L D L fraction was delipidated sequentially, first with 10 ml of an acetone:ethanol mixture (1:1, v/v) which was then stored overnight at -20°C. Subsequently, the sample was centrifuged at 1,500 x g for 15 min. and the solvent discarded. Another 10 ml of the acetone:ethanol mixture was added to the pellet which was vortexed and stored at -20°C for 2 hr. After centrifugation, the supernatant was removed and a final delipidation step was performed. Five ml of diethyl ether were added to the pellet which was vortexed and stored at -20°C for lhr. After centrifugation, the solvent was discarded and the residual ether was evaporated so that only the dry apoprotein residue remained. On the day of the focusing experiment, the apo V L D L was solubilized in 200 /d of 0.01M Tris, 0.02M dithiothreitol and 8M urea solution, p H 8.2. 2.4.2 Isoelectric focusing For 10 ml of gel solution, 0.75 g of acrylamide, 20 mg of bisacrylamide, 4.8 g of urea and 0.5 ml of p H 4-6 ampholines were dissolved and made to the final volume with distilled water. Ten ul of T E M E D and 40 /d of 10% (w/v) ammonium persulfate (made fresh) were added to the gel solution which was poured into tubes (100mm x 5mm) sealed with parafilm. The gel is overlayed with water to form a smooth interface and allowed to polymerize for 30 min. Once polymerized, the gel tubes are loaded into the electrophoretic chamber with a lower reservoir buffer of 19 lOmM H 3 P 0 4 and an upper reservoir buffer of 20mM NaOH. The gels are pre-focused at a constant voltage of 110 volts for 1 hr. After pre-focusing, the samples are loaded and overlayed with 200 pi of diluted (two-fold) sample buffer containing 1% ampholines. The gel tubes are focused for 16 hours at 250 volts constant voltage with running water as a coolant. A final focus at 450 volts for 1 hr. is performed before removing the gels with a 22 gauge spinal needle and water spray. 2.4.3 Gel staining A stock staining solution consisting of 0.4 g of Coomassie Blue G250 in 1000 ml of 3.5% (v/v) perchloric acid was used. Ten ml of stain was added to each tube containing a gel and was rotated end-over-end for 2hr. After staining was completed, the solution was decanted and replaced with destain (7.5% acetic acid) and rotated overnight or until background was clear. Finally, the gels were scanned with a Beckman Appraise 1123 densitometer. 2.5 Apo E Phenotyping directly from plasma 2.5.1 Sample preparation Ten fil of plasma or serum was mixed with 40 ul neuraminidase (0.001U//d) in 0.02M sodium acetate buffer p H 5.1 in a 1.5 ml microcentrifuge tube and incubated at 37°C for 30 min. Following the incubation the mixture was treated with 1.0 ml ethanol/diethyl ether (3:1, v/v) at -20°C. After vortexing, the precipitate was immediately centrifuged at 10,000 x g for 10 min. After removal of the supernatant, the pellet was subsequently washed with diethyl ether at -20°C, vortexed and rocked for 15 min. After centrifugation, the supernatant was aspirated off and the pellet was allowed to air dry at which point it could be stored at -20°C. The protein pellet was dissolved in 100 pi of 8M urea containing 2% Nonidet P-40, 5% 6-mercaptoethanol 20 and 0.8% ampholines (pH 5-6.5). The resolubilized protein was allowed to incubate at room temperature for 30 min. and was then revortexed before 15 ul were loaded onto the IEF gel. 2.5.2 Isoelectric focusing Two mini-vertical slab gels (60mm x 90mm) for IEF were prepared and run simultaneously using a BioRad Mini-Protean II system. The gel solution contained 5% acrylamide, 0.43% N,N'-methylenebisacrylamide, 2.5% ampholines (pH 5-6.5), 8M urea and 0.1% Nonidet P-40. After the additions of 60 ul of 10% (w/v) ammonium persulfate (made fresh) and 15 pi T E M E D to 15 ml of gel solution (enough for two gels), the mixture was poured between two glass plates separated by 0.75mm spacers. Two 15 well templates were inserted between each set of glass plates and the gels were allowed to polymerize (30-45min.). Once polymerization was complete, the templates were removed and the wells were rinsed with distilled water. The cationic electrophoresis buffer was lOmM H 3 P 0 4 and the anionic buffer solution was 20mM N a O H . Samples were underlayed below an overlay buffer containing 4M urea, 1% ampholines (pH 5-6.5) and 0.025% (w/v) bromphenol blue which was diluted 1:1 with distilled water before application. Gels were run at a constant current of 2.5 mA/gel with no cooling required and focusing was determined to be complete when the voltage approached a constant level of approximately 650 volts. Routinely, this required 2hr. of focusing time. 2.5.3 Immunoblotting Following completion of isoelectric focusing, the gels were removed from the glass plates and rinsed briefly in blotting buffer (0.7% acetic acid). Before application to the gel, nitrocellulose paper (65mm x 95mm) of a 0.45 /jm pore size was placed in 21 hot water to ensure complete wetting of the membrane. Electroblotting was carried out at a constant voltage of 100 volts with a starting current of 310 mA for 1 hour. After blotting, the nitrocellulose membrane was incubated at 37°C and shaken for 30 min. in TBS buffer (150mM NaCl, 20mM Tris, pH7.4) containing 5% (w/v) nonfat dry milk to block remaining protein binding sites. The blot was then incubated at room temperature in 10 ml of TBS buffer containing 0.5% (w/v) nonfat dry milk and 10 u\ (1:1000 dilution) of goat-anti-human apo E polyclonal antiserum. After 1 hour incubation, the blot was washed four times for 5 min. in 20 ml of TBS buffer containing 0.02% Tween 20. Detection of the bound anti-apo E antibody was achieved by an incubation at room temperature in 10 ml of TBS buffer containing 0.5% nonfat dry milk and 5 /zl (1:2000 dilution) of Protein G conjugated to horseradish peroxidase. After 30 min., the blot was again washed four times in TBS buffer containing 0.02% Tween 20. To visualize the focused bands the blots were developed at room temperature in a substrate solution containing 50 mg 3,3-diaminobenzidine tetrahydrochloride (DAB), 30 mg CoCl 2 and 20 /il of 30% (w/v) H 2 0 2 dissolved in 100 ml of TBS buffer. The enzyme reaction was stopped when visualization was complete by washing the blot with distilled water. 2.6 Data Analysis Statistical analysis was performed using data obtained on the first visit of the patient to the lipid clinic. Patients with secondary causes of hypercholesterolemia and those on medication affecting lipoprotein metabolism were excluded from the analysis. The significance of difference between two means was determined by the students Mest. The statistical significance of the differences in proportion between 22 two groups was determined by the chi-square test (employing Yates correction for continuity). The average effects for each apo E allele on lipid and apoprotein levels were estimated according to the formula of Sing and Davignon (57). 23 3 RESULTS 3.1 Apo E Phenotyping 3.1.1 Neuraminidase treatment Figure 4 shows a typical immunoblot of the isoelectric focusing patterns of the six common apo E phenotypes with and without neuraminidase pretreatment of plasma. The enzymatic removal of the sialic acid residues in plasma prior to delipidation greatly reduced the intensity of the sialylated isoforms thereby clarifying the specific B 1 2 3 4 5 6 A B C D E F Figure 4. Apo E immunoblots depicting the six common phenotypes. A - untreated, B - pretreated with neuraminidase. 24 3.1.2 Comparison of traditional and new phenotyping methodologies We compared the results of the immunoblotting method with the traditional V L D L / I E F procedure by using the same plasma obtained from 51 patients attending the University Hospital Lipid Clinic. Four of these samples could not be assigned a phenotype with the V L D L / I E F procedure as there was an insufficient amount of apo E to be visualized. This is a common difficulty with normotriglyceridemic specimens. However, this was not a problem using the IEF/immunoblot method since it relies on the increased sensitivity of an antibody detection system. A n independent skilled technician with experience in the V L D L / I E F procedure attempted to classify the remaining 47 samples (Table 1). The band pattern for two of these samples remained ambiguous as the phenotypes E3/3 and E3/2 could not always be clearly distinguished. Both of these samples were clearly identified as the E3/3 phenotype by our immunoblot method. With the exception of one, the remaining samples were in agreement for both techniques. The single mismatch was assigned as E3/2 by the V L D L / I E F method but was clearly demonstrated as an E3/3 pattern when visualized on the immunoblot. 25 Table 1. Apolipoprotein E phenotype assignments using two different methodologies. No. Identified Phenotype V L D L / I E F Plasma/IEF/Immunoblot E4/4 2 2 E4/3 12 12 E4/2 1 1 E3/3 18 21 E3/2 6 5 E2/2 6 6 Ambiguous 2 0 Total 47 47 3.1.3 Allele frequencies in a population from Vancouver The new methodology was subsequently applied to phenotype 203 subjects from Vancouver. The phenotype distribution and allele frequency are displayed in Table 2, The frequency of the six phenotypes were 1.4% for E4/4, 24.6% for E4/3, 3.0% for E4/2, 57.6% for E3/3, 12.3% for E3/2, and 1.0% for E2/2 which remained consistent in both sexes. The apo E allele frequencies were 0.153 for e4, 0.761 for e3 and 0.086 for el. 26 Table 2. Apolipoprotein E phenotype and allele frequencies in a normal population from Vancouver. Phenotype N o . Percent E 4 / 4 3 1.4 E 4 / 3 50 24.6 E 4 / 2 6 3.0 E 3 / 3 117 57.6 E 3 / 2 25 12.3 E 2 / 2 2 1.0 T O T A L 203 100 A l l e l e Frequency e4 = 0.153 e3 = 0.761 e2 = 0.086 3.1.4 Apo E phenotype and lipid profile The l ip id , l ipoprotein and apoprotein profiles for the six different phenotypes are shown i n Table 3. Individuals having phenotypes containing the e2 allele ( E 4 / 2 ; E 3 / 2 ; E 2 / 2 ) had significantly lower levels of total cholesterol (P < 0.01), L D L cholesterol (P < 0.001) and apoprotein B (P < 0.001) than those without it. In contrast, phenotypes containing the e4 allele ( E 4 / 4 ; E 4 / 3 ; E 4 / 2 ) had higher levels of these same three parameters than those not having this gene but these differences 27 Table 3. Apolipoprotein E phenotype and levels of lipids, lipoproteins and apoproteins. TOTAL = 203 Apo E Phenotype 4/4 4/3 3/3 4/2 3/2 2/2 (n) (3) (50) (117) (6) (25) (2) Age 36.3±11 45.9±17 42.9118 47.0119 41.0116 30.519 Total Cholesterol (mmol/L) 5.30±0.7 5.53±1.0 5.2911.2 4.8311.0 4.8811.2 3.8610.4 LDL Cholesterol (mmol/L) 3.73±0.6 3.57±0.9 3.4911.0 2.8910.8 2.8810.6 2.2010.1 HDL Cholesterol (mmol/L) 1.14±0.1 1.3310.4 1.2610.3 1.2310.4 1.3810.2 1.1710.03 Triglycerides (mmol/L) 0.95±0.3 1.2810.8 1.3110.8 1.5510.6 1.4511.0 1.0810.4 Apoprotein B (g/L) 0.91±0.2 0.8910.2 0.8610.2 0.7510.2 0.7210.3 0.4110.01 Apoprotein A-I (g/L) 1.34±0.1 1.5110.3 1.4310.3 1.4210.4 1.5310.2 1.4810.1 Values are given as means 1 standard deviations except for E2/2 where the data is expressed as the mean 1 h the range. 28 did not reach statistical significance. There was no evidence in this population of a significant variation between apo E phenotypes and the mean levels of triglycerides, H D L cholesterol or apoprotein A-I. Table 4 summarizes the estimates of the average effects of each allele for each of the parameters analysed. The effect of the e2 allele in decreasing total cholesterol, L D L cholesterol and apoprotein B was clearly apparent. This allele was also associated with a considerable increase in triglycerides and a modest increase in H D L cholesterol and apoprotein A-I. As expected, the e3 allele did not appear to have any influence on these variables. Although considerably less dramatic, the e4 allele appeared to have the opposite effect of the e2 allele. This allele was associated with moderate increases in total cholesterol, L D L cholesterol and apoprotein B and with a decrease in triglycerides. Table 4. Estimation of the average effects of the three apo E alleles on the levels of lipids, lipoproteins and apoproteins, expressed as a percentage of the respective total population means. Apo E Allele e2 e3 c4 Frequency 0.086 0.761 0.153 Total Cholesterol -9.3 0.4 2.5 L D L Cholesterol -17.3 1.2 2.5 H D L Cholesterol 3.7 -0.8 0.3 Triglycerides 8.5 -0.3 -3.9 Apoprotein B -17.5 1.5 3.8 Apoprotein A-I 3.7 -0.7 1.1 29 3.2 F H Population 3.2.1 Age and sex distribution A total of 364 patients (208 women and 156 men) with F H were identified from 283 families. The age distribution of this population is shown i n Figure 1. The mean age for men was 40 .3±17 years and 45.4±17 years for women. This age difference was statistically significant (P < 0.01) with a larger proportion of females found between the ages of 50 and 69. 5 0 - . 4 5 -4 0 -3 5 -3 0 -2 5 -2 0 -1 5 -1 0 -5 -0 -Q. D p O < n -*-* c .2 o Q_ o Z 50 - i 4 5 -4 0 -35 3 0 -2 5 -2 0 -1 5 -1 0 -5 -0 id id \d FEMALES (n=208) 0-9 10-19 20-29 30-39 40-49 50-59 80-69 70-79 id id y r y s y * > y MALES (n=156) _0_ 0-9 10-19 20-29 30-39 40-49 50-59 60-89 70-79 Age in Years Figure 5. Age distribution of F H patients for each sex. 30 3.2.2 Plasma lipids, lipoproteins and apoproteins Table 5 compares the lipid parameters between men and women with F H and contrasts this patient group with the randomly selected population. As expected, in both sexes the levels of total cholesterol, L D L cholesterol and apoprotein B were greatly elevated in comparison to the normal population. In addition, the levels of H D L cholesterol and apoprotein A-I were consistently lower in all F H patients when compared to normals (P < 0.001). Interestingly, the levels of triglycerides were significantly elevated in F H females in comparison to normal females (P < 0.005). Also, Table 5 shows several differences among these variables between males and females with F H . The levels of total, L D L and H D L cholesterol were all significantly elevated in F H women as compared to the F H males. The mean cholesterol value for women was 9.05±1.7 mmol/L and for men 8.48±1.5 mmol/L (P < 0.001). The magnitude of this difference was reflected in L D L cholesterol, 6.99±1.6 mmol/L for females and 6.63±1.5 mmol/L for males (P < 0.05) and H D L cholesterol, 1.26±0.3 mmol/L for females and 1.09±0.2 mmol/L for males (P < 0.001). The levels of plasma triglycerides in this F H population did not differ significantly between the sexes. However, there was a significantly greater concentration of apoprotein B and apoprotein A-I in females compared to males (P < 0.001). The concentration of lipids and apoproteins was also analysed for each age group within each sex (Table 6). The values of total and L D L cholesterol increased steadily with age in both sexes. Higher levels of total cholesterol were observed in females for each age group. The concentration of H D L cholesterol after age 30 appeared to decline for males but increase for females with lower values found in males for the 31 remaining age groups. Although there was no significant difference between triglyceride values between the sexes in the whole group, differences could be detected when the separate age groups were analysed. For males, the concentration of triglycerides increased with age and peaked between the ages of 30 and 49 and then declined after age 50. In contrast, females generally showed a steady increase in the concentration of triglycerides with age. Between the ages of 30 and 49, males had Table 5. Lipids, lipoproteins and apoproteins in FH patients. MALES FEMALES NORMAL (97) FH (156) NORMAL (101) FH (208) Age 44.5±18 40.3±17 41.8±17 45.4±17+ Total Cholesterol (mmol/L) 5.37±1.1 8.48±1.5* 5.26±1.2 9.05±1.7*+ L D L Cholesterol (mmol/L) 3.50±0.9 6.63±1.5* 3.30±1.1 6.99±1.6*+ H D L Cholesterol (mmol/L) 1.20±0.3 1.09±0.2* 1.40±0.3+ 1.26±0.3*+ Triglycerides (mmol/L) 1.45±0.8 1.53±0.9 1.20±0.8+ 1.48±0.8* Apoprotein B (g/L) 0.89±0.2 1.35±0.5* 0.81±0.2+ 1.52±0.4*+ Apoprotein A-I (g/L) 1.42±0.3 1.24±0.2* 1.55±0.3+ 1.36±0.3*+ Values are given as means ± standard deviations. * The significance of difference between normal and F H groups (P < 0.001). •f The significance of difference between males and females with F H (P < 0.05). 4= The significance of difference between males and females in the normal population (P < 0.05). 32 Table 6. Mean values for lipids, lipoproteins and apoproteins for each age division in FIT. MALES (TOTAL = 156) 0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 (12) (9) (19) (28) (35) (32) (19) (2) Total Cholesterol (mmol/L) 7.24 + 1.5 7.6311.5 8.5411.3 8.4911.3 8.8111.6 8.6311.2 8.8411.7 6.9110.1 L D L Cholesterol (mmol/L) 5.66±1.4 5.7711.4 6.9011.3 6.3911.6 6.97+1.6 6.8811.2 6.8611.4 5.3910.4 HDL Cholesterol (mmol/L) 1.14±0.2 1.1610.2 1.11±0.2 1.0910.2 1.0410.3 1.0510.2 1.1510.2 0.9210.1 Triglycerides (mmol/L) 0.99±0.4 0.7510.3 1.2010.7 1.8811.2 1.8410.8 1.5710.7 1.4510.7 1.4310.4 Apoprotein B (g/L) 1.1510.3 1.0910.2 1.3010.4 1.3510.4 1.37+0.7 1.5010.3 1.3810.4 1.4110.1 Apoprotein A-I (g/L) 1.22±0.1 1.3010.2 1.1610.1 1.2410.2 1.1810.3 1.2510.2 1.3410.2 1.3110.03 FEMALES (TOTAL = 208) 0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 (9) (8) (18) (44) (30) (45) (45) (9) Total Cholesterol (mmol/L) 7.65±0.9 8.5611.4 8.5711.4 8.9611.9 9.0011.3 9.2112.0 9.4811.5 9.3912.0 L D L Cholesterol (mmol/L) 6.11 ±0.8 6.8511.2 6.8511.4 7.0111.8 6.9111.4 6.9611.7 7.3311.6 6.90+2.0 HDL Cholesterol (mmol/L) 1.09±0.2 1.0610.2 1.2210.3 1.2010.3 1.4110.4 1.2810.4 1.2410.3 1.4210.3 Triglycerides (mmol/L) 1.07±0.2 1.4110.7 1.0410.4 1.2410.7 1.2911.0 1.6110.7 1.8610.9 2.2011.0 Apoprotein B (g/L) - 1.28±0.2 1.5310.4 1.4810.5 1.4810.5 1.6110.4 1.5210.3 1.5910.4 1.4710.4 Apoprotein A-I (g/L) l.I9±0.1 1.2410.2 1.2510.3 1.2810.3 1.4010.3 1.4410.3 1.4110.3 1.3910.3 Values are given as means 1 standard deviations except for the male age group of 70-79 where the data is expressed as the mean 1 h the range. 33 significantly higher triglyceride levels than females, 1.85±1.0 mmol/L compared to 1.26±0.9 mmol/L (P < 0.001). The values of apo B for all ages were consistently higher in females. In contrast, apo A-I levels were similar for both sexes until the age of 40 years at which point it increased significantly in females (P < 0.002). 3.2.3 Clinical Features o f F H The frequency of specific clinical findings in F H for each age group is listed in Table 7. Tendon xanthomas began to appear most commonly after age 20 and their frequency increased with age. The total incidence of tendon xanthomas was greater in females (89%) compared to males (77%) (P < 0.01). The frequency of corneal arcus also increased with age but was more common in men (60%) than women (46%) (P < 0.05). After age 30, the clinical findings related to coronary artery disease first appeared and became more prevalent with increasing age. The occurrence of MI was much greater in men over 30 (27%) than women of the same age (7%)(P < 0.001). Angina was also more frequent in males (18%) than females (9%) for this age group (P< 0.05). Stroke or cerebrovascular disease occurred in only 1 male and 6 females between the ages of 50 and 69. The cumulative frequency of C A D in F H as a function of age is shown in Figure 6. Males had a much higher incidence of disease with a frequency of 31% for the total male population as compared to 13% for females (P < 0.001). Figure 7 depicts the distribution of the age of onset of symptoms of coronary disease in F H . For males, symptoms occurred from as early as ages 20 to 29 but the most common age of onset was between the ages of 40 and 49 where the frequency of disease was greater than 50%. In contrast, for females, the earliest symptoms of C A D occurred 34 Table 7. Clinical data for each age division in M l . MALES (TOTAL = 156) 0-19 20-29 30-39 40-49 50-59 60-69 70-79 Clinical Finding (21) (19) (28) (35) (32) (19) (2) Tendon Xanthoma 0 12 (63%) 25 (89%) 32 (91%) 31 (97%) 18 (95%) 2 (100%) Corneal Arcus 1 (5%) 7 (37%) 11 (39%) 31 (89%) 25 (78%) 17'(89%) 2 (100%) Myocardial Infarction 0 0 5 (18%) 6 (17%) 11 (34%) 8 (42%) 1 (50%) Angina 0 0 2 (7%) 6 (17%) 6 (19%) 5 (26%) 2 (100%) Bypass Surgery 0 0 0 7 (20%) 9 (28%) 3 (16%) 1 (50%) Angiographic Disease 0 0 2 (7%) 11 (31%) 5 (16%) 3 (16%) 2 (100%) Stroke 0 0 0 0 1 (3%) 0 0 F E M A L E S (TOTAL = = 208) 0-19 20-29 30-39 40-49 50-59 60-69 70-79 Clinical Finding (17) (18) (44) (30) (45) (45) (9) Tendon Xanthoma 3 (18%) 14 (78%) 40 (91%) 29 (97%) 45 (100%) 45 (100%) 9 (100%) Corneal Arcus 1 (6%) 3 (17%) 16 (36%) 13 (43%) 31 (69%) 27 (60%) 5 (56%) Myocardial Infarction 0 0 2 (5%) 1 (3%) 5 (11%) 4 (9%) 0 Angina 0 0 1 (2%) 1 (3%) 2 (4%) 8 (18%) 3 (33%) Bypass Surgery 0 0 1 (2%) 2 (7%) 3 (7%) 2 (4%) 0 Angiographic Disease 0 0 2 (5%) 2 (7%) 0 2 (4%) 0 Stroke 0 0 0 0 4 (9%) 2 (4%) 0 Values are given as absolute numbers and as percentages in parentheses of the total number for each age group. 35 males, symptoms occurred from as early as ages 20 to 29 but the most common age of onset was between the ages of 40 and 49 where the frequency of disease was greater than 50%. In contrast, for females, the earliest symptoms of C A D occurred between the ages of 30 and 39 after which the incidence of disease increased with age. The mean age of onset was 48.2±9 years for men and 54.7±12 years for women (P < 0.05). 40 n o 30 • c CD D c r CD £ 20 CD > ZJ £ ZJ O 10-r-0 10 "20 MALES FEMALES —1— 30 40 50 Age Divisions 60" 70 Figure 6. The cumulative frequency of C A D in males and females with F H . 36 60-, < 50 O :§ 40 cn 30-£ 20-o 10H 0 CZL MALES FEMALES Ll 0-19 20-29 30-39 40-49 50-59 60-89 70-79 Age in Years Figure 7. The distribution of the age of onset of symptoms of CAD in F H . 3.2.4 Plasma lipids, lipoproteins and apoproteins for those with and without CAD To assess differences associated with the presence or absence of CAD, only those F H patients > 30 years of age were analysed. Table 8 splits the men and women populations into two groups and compares the same lipid parameters for those with or without coronary disease. The concentration of L D L cholesterol was higher in men with CAD, 7.13±1.5 mmol/L compared to 6.5J+1.4 mmol/L (P < 0.05). The values for H D L cholesterol for CAD positive males (0.99±0.2 mmol/L) was significantly lower than the CAD negative group (1.13±0.2 mmol/L)(P < 0.001). The 37 difference observed for these values was also reflected in significantly higher apo B and lower apo A-I levels for males with CAD. In contrast, the only difference seen between CAD positive and negative females was the level of triglycerides which were significantly elevated in women with CAD, 2.09±1.4 mmol/L compared to 1.46±0.7 mmol/L (P < 0.001). Table 8. Lipids, lipoproteins and apoproteins in FH patients with and without CAD. MALES FEMALES CAD (•) CAD ( + ) CAD (-) CAD ( + ) n 68 47 147 26 Age 45.5±10 48.2±9 49.7±12 54.7±12 Total Cholesterol (mmol/L) 8.47±1.3 8.96±1.6 9.10±1.6 9.68±2.1 LDL Cholesterol (mmol/L) 6.51±1.4 7.13±1.5* 7.01±1.6 7.25±2.0 HDL Cholesterol (mmol/L) 1.13±0.2 0.99±0.2*** 1.29±0.3 1.22±0.4 Triglycerides (mmol/L) 1.70±1.0 1.70±0.7 1.46±0.7 2.09±1.4*** Apoprotein B (g/L) 1.33±0.4 1.51±0.5* 1.53±0.4 1.59±0.6 Apoprotein A-I (g/L) 1.28±0.2 1.1810.2** 1.40±0.3 1.31±0.3 Values are given as means ± standard deviations. The significance of difference between CAD ( + ) and CAD (-) groups for males and females is P < 0.05*, P < 0.01**, P < 0.001***. 3.2.5 Frequency of risk factors for the CAD ( + ) and CAD (-) groups The frequencies of common risk factors for the CAD positive and negative 38 groups are compared in Table 9. Smoking was associated with the presence of C A D in males (P < 0.005) but not in females. However, a higher frequency of hypertension was observed in women with C A D but not among men (P < 0.025). A higher percentage of men with C A D had H D L cholesterol levels in the lower quartile of this population. (P < 0.025). Conversely, significantly fewer males with H D L cholesterol levels in the upper quartile were positive for C A D (P < 0.025). Similar trends were observed for females but these differences were not statistically significant. There was no difference in body mass index (BMI) between those with and without C A D for both sexes. Table 9. Assessment of the effects of smoking, hypertension, H D L cholesterol levels and BMI on CAD in F H . M A L E S F E M A L E S CAD (•) CAD ( + ) CAD (-) CAD ( + ) n 68 47 147 26 Smoking 26 (38%) 33 (70%)** 58 (39%) 9 (35%) Hypertension 7 (10%) 3 (6%) 18 (12%) 9 (35%)* H D L Cholesterol in lower quartile 11 (16%) 18 (38%)* 34 (23%) 9 (35%) H D L Cholesterol in upper quartile 23 (34%) 6 (13%)* 38 (26%) 5 (19%) BMI 24.75±3.0 25.15±2.8 24.10±4.0 25.31±3.5 The significance of difference between C A D (+) and C A D (-) groups for males and females \sP < 0.05*, P < 0.01**. 39 3.3.6 Apolipoprotein E and FH The apo E phenotype distribution and allele frequency for both F H and normal populations are shown in Table 10. Although not statistically significant, the frequency of the E4/3 phenotype was higher in F H patients (33.6%) compared to normals (24.6%) while the E3/2 incidence was lower, 8.0% compared to 12.3%. These differences were also reflected for the individual allele frequencies. In F H they were 0.056 for el, 0.744 for e3, 0.200 for e4 and in normals, 0.086 fore2, 0.761 for e3 and 0.153 for e4. Table 10. Apo E phenotype distribution and allele frequency in FH. P O P U L A T I O N % F H N O R M A L (125) (203) Apo E Phenotype: E4/4 1.6 1.4 E4/3 33.6 24.6 E4/2 3.2 3.0 E3/3 53.6 57.6 E3/2 8.0 12.3 E2/2 0.0 1.0 Apo E Allele: e4 £3 a 0.200 0.744 0.056 0.153 0.761 0.086 This F H population was separated into two sets of two groups; those who had a phenotype containing the apo E4 or the apo E2 isoform and those who did not. 40 Clinical data for these patients were compared in Table 11. The incidence of tendon xanthomas, corneal arcus, smoking and hypertension remained consistent for all groups. The only observable difference was found in the frequency of C A D , 29% for those with E4 and 19% for those without, but this difference did not reach statistical significance. It should be noted that there was no difference between these patients with C A D with respect to mean age, sex distribution or the frequency of smoking and hypertension (data not shown). Table 12 displays the lipid and lipoprotein data for the same categories where the only significant difference found was a distinct elevation of triglycerides associated with the E2 isoform (P < 0.001). It should be recognized that the ratio of men to women in each group was similar and that a separate analysis for males and females revealed the same relationships observed in Table 12 (data not shown). Table 11. Clinical data in FH patients with and without apolipoprotein E4 and E2. E4 (-) E4 ( + ) E2 (-) E2 ( + ) n 77 48 111 14 Tendon Xanthomas 71 (92%) 43 (90%) 101 (91%) 13 (93%) Corneal Arcus 48 (62%) 26 (54%) 66 (59%) 8 (57%) Smoking 27 (35%) 16 (33%) 36 (32%) 7 (50%) Hypertension 11 (14%) 6 (13%) 16 (14%) 1 (7%) Coronary Artery Disease 15 (19%) 14 (29%) 26 (23%) 3 (21%) 41 Table 12. Lipid, lipoprotein and apoprotein levels in FH patients with and without apolipoprotein E4 and E2. E4(-) E4( + ) E2(-) E2( + ) n 77 48 111 14 Age 45.6±17 47.7±14 46.6±16 44.5±12 Total Cholesterol (mmol/L) 9.32±1.9 8.81±1.7 9.07±1.7 9.56±2.5 L D L Cholesterol (mmol/L) 7.16±1.9 6.54±1.7 6.97±1.7 6.52±2.4 H D L Cholesterol (mmol/L) 1.13±0.3 1.18±0.3 1.16±0.3 1.08±0.2 Triglycerides (mmol/L) 1.75±0.9 1.79±1.2 1.64±0.9 2.70±1.5* Apoprotein B (g/L) 1.53±0.5 1.46±0.5 1.50±0.5 1.47±0.4 Apoprotein A-I (g/L) 1.28±0.3 1.31±0.2 1.28±0.3 1.35±0.2 Values are given as means ± standard deviations. The significance of difference between the ( + ) and (-) groups for each apo E isoform is P < 0.001*. 42 4 D I S C U S S I O N 4.1 Apo E Phenotyping Methodology The immunoblot method for apo E phenotyping described i n this report offers many advantages over traditional procedures. It uses a small sample volume of serum or plasma (10/zl), obviates the need for ultracentrifugation to separate the V L D L fraction, and allows a greater number of samples to be processed at the same time. In addition, the use of frozen plasma, the simplification of the sample treatment, and the ability to run 60 samples simultaneously makes this method useful for population screening. The removal of the sialic acid residues from apo E in plasma with neuraminidase has eliminated additional bands which may interfere with the correct assignment of apo E phenotype. Consequently, the clarity of the final band patterns is a considerable benefit as it avoids the confusion of evaluating intensity ratios of various bands associated with the V L D L / I E F method. Recently, a similar procedure has been described using an agarose gel medium (88). However, i n our laboratory, we have found that using acrylamide minigels offers several advantages over this method. The smaller volumes of solutions required throughout this technique are an added convenience and provide a significant savings in reagent costs. Af ter a short time of polymerization, acrylamide gels can be used immediately unlike agarose gels which require an extended period of t ime to set. In addition, the acrylamide I E F gels do not require a pre-focusing step, can focus in a shorter time and do not require a cooling mechanism. Final ly, the detection system consisting of a one hour first antibody incubation followed by only a 30 minute incubation with Protein G increases the efficiency over other methods previously 43 described. The accuracy of the immunoblotting method was confirmed by comparing the phenotypes obtained to those identified by the isoelectric focusing of delipidated V L D L . In addition, this procedure was able to definitively assign phenotypes to the 12% of the samples studied which could not be identified by the existing method. In summary, the low cost, speed and simplicity of this procedure makes it ideal for clinical laboratory applications and for screening a variety of large populations. This new phenotyping procedure was applied to a large randomly selected population from Vancouver to determine the local distribution of the apo E alleles as differences in allele frequencies have been observed between different populations (56). This cohort comprised of approximately 10% non-Caucasian, most of which were Asian, with the remaining majority being Caucasian. The apo E allele frequencies of this population (e4 = 0.153, e3 = 0.761, e2 = 0.086) were almost identical to those observed in a second Canadian population from Ottawa (e4 = 0.152, e3 = 0.770, e2 = 0.078) (57). These data are compatible with studies from a variety of Caucasian populations which have similar allele frequencies (75) confirming the validity of our method. The contrasting allelic effects on the concentration of lipids and apoproteins in this population were consistent with previous observations (75). Individuals with phenotypes containing the E2 isoform had lower levels of total cholesterol, L D L cholesterol and apoprotein B. Although the presence of the E4 isoform was associated with a greater concentration of these parameters, the "cholesterol-lowering" effect of the e2 allele is usually two to three times the "cholesterol-raising" effect of e4 (75). At this level of analysis, there was no apparent relationship between 44 apo E phenotype and the concentration of triglycerides, H D L cholesterol or apoprotein A-I. However, determining the influence of the different alleles is better assessed through a calculation estimating the average effect of each allele (57) (Table 4). From this analysis, it was evident that the opposing effects of the el and e4 alleles on cholesterol levels could also be extended to the concentration of triglycerides. In this case, the el allele was identified with a considerable elevation in triglycerides while the effect of e4 was to moderately decrease triglyceride levels. These observations can be explained by the same mechanism proposed to describe the effect of apo E on serum cholesterol levels. As outlined earlier, there are differences in the catabolic efficiencies between the E2 and E4 isoforms. In addition to modifying L D L levels, the metabolism of chylomicron and V L D L remnants is affected directly by apo E . As a result, it is likely that the triglyceride component of these lipoproteins would contribute to the differences in plasma triglycerides related to the two isoforms. From this study and others, it is apparent that the impact of allelic variation at the apo E locus in a normal population is visible in the altered values of cholesterol, triglycerides and apoprotein B. 4.2 F H Population The detailed analysis of such a large population of patients with heterozygous F H revealed a relatively low incidence of C A D in the presence of a gender-specific response to selected risk factors. The higher proportion of females identified with F H is most likely due to the earlier death from C A D in male heterozygotes (89). This observation would also explain the greater mean age associated with the female population. However, it should be noted that in several cases the clinical and 45 laboratory data presented here represents patients who later died during the time period after the initial visit to the Lipid Clinic and the completion of the study. As reported previously (12,28-30), the concentration of H D L cholesterol was significantly lower in both sexes compared to the mean levels in a normal population. In addition, the levels of apo A-I paralleled those of H D L cholesterol and were consistently lower in all F H patients. Although the reason for these decreases are not clear, it appears that the metabolism of H D L is in some way affected by a defect in the catabolism of LDL. It is possible that the altered kinetics of these lipoproteins including IDL may also influence the metabolism of plasma triglycerides. For example, in this study, women with F H had elevated triglycerides when compared to age-matched normals. It was also of interest that both H D L cholesterol and triglycerides appeared to have a better predictive value for the development of coronary disease than either total or L D L cholesterol. When the lipid and lipoprotein data of males and females with F H were analysed, several differences were observed. For the first time it has been shown that women with F H have significantly higher mean levels of total and L D L cholesterol. This observation did not appear to be influenced by an age difference as the total cholesterol levels were higher in every age group. In addition, this finding correlated well with the higher frequency of tendon xanthomas in females, especially those aged less than 30 (Table 3). Given the selection criteria, these characteristics may have also contributed to the greater number of females seen in this F H population. The sex related differences of H D L cholesterol in the normal population are still evident in FH. In fact, higher values in F H women may be one of the reasons for their 46 decreased susceptibility to C A D . In contrast, differences in triglyceride values were seen only between specific age groups. As in the general population, higher levels of triglyceride were observed in males between the ages of 30 and 49. The reported incidence of C A D of 31% for men and 13% for women was somewhat lower compared to other studies (28,33,34); however this may have been affected by the lower mean age of this population compared to the average age associated with the first symptoms of C A D . The age of onset of coronary symptoms and its delay for females, 55 years compared to 48 years for males, has been shown to be a remarkably consistent feature in several different F H populations (28,32,35,36). Another possible explanation for the low incidence of C A D in this cohort may be due to differences in the genetic background of the population. The diversity in ethnic origin of these subjects would indicate that a large degree of heterogeneity exists in the characteristics of the genetic mutation. In contrast, the studies mentioned previously (28,33,34) are likely to be more homogeneous with respect to genetic mutation as they consisted of French Canadians (28), 11 Danish families (33) and a small Japanese population (34). In this study, it appears that the lack of genetic uniformity has resulted in a generally less severe clinical expression of F H . The levels of total cholesterol in either sex were not able to distinguish those patients with coronary disease. This was particularly evident in women who had higher plasma cholesterol levels than men but still had a very low incidence of C A D . However, for men, the concentration of both L D L and H D L cholesterol had a greater predictive value. This observation was also confirmed in the measurement of 47 apoprotein concentration with lower values of apo A-I and higher values of apo B being more often associated with C A D . Although other reports have shown that low H D L cholesterol in F H was associated with C A D in both sexes (30,34), our study found that it was a good indicator for males only. In contrast, the only difference seen for females was higher concentrations of triglyceride for those positive for C A D , an observation also made by Hirobe et al. (34). In addition, the elevation in triglycerides was independent of BMI values which were similar in all groups (Table 5). The differences between the sexes still remained visible after additional risk factors were assessed. Smoking was found to have a profound effect in men where as much as 70% of the C A D positive males were smokers. A n increased risk of coronary disease has been associated with smoking in other F H studies (32,37) especially for males (32) but it remains unclear why female smokers are not similarly affected. Instead, hypertension, which was seen with greater frequency in women was associated with coronary disease. Having been reported in other studies (32,35), this observation suggests that hypertension is an independent risk factor for females with F H . When H D L cholesterol levels were divided into quartiles, the relationship between low values and C A D was confirmed in males. Also, it was interesting to note that a higher percentage of patients with an H D L cholesterol value in the upper quartile were negative for C A D . This observation, also reported by Hirobe et al. (34), suggests that higher levels of H D L cholesterol offer a degree of protection against coronary disease even in the presence of hypercholesterolemia. 48 The analysis of the apo E polymorphism in 125 unrelated F H subjects revealed several similarities to the study of Eto et al. of 50 Japanese patients with F H (80). In their study, there was also a tendency of the e4 allele to have a higher frequency in F H compared to the normal population. In addition, the prevalence of ischemic heart disease was greater in those patients which were positive for apo E4 while the frequency of the other risk factors did not differ between the two groups. However, an important difference in our study, as well as others (81-83), was the lack of a relationship between apo E4 and the concentration of any of the parameters in the plasma lipid profile. From this observation, it is difficult to explain an increase in the frequency of the e4 allele in F H and its propensity to be related with C A D . In addition, although the locus for both the apo E and L D L receptor genes is found on chromosome 19 (25), their distant locations would not indicate a functional relationship (90). It is likely that in most cases a mutation in the L D L receptor gene would affect cholesterol metabolism to such an extent that the influence of the different apo E alleles on cholesterol concentration seen in the normal population would not be evident in those with F H . However, it is of interest that those patients who have phenotypes containing the E2 isoform have elevated triglycerides, an observation sometimes seen in the normal population (91,92). Yet, it appears that the triglyceride raising effect associated with the E2 isoform is much more dramatic in F H (83). As a result, the presence of the E2 isoform may be associated with an increased risk of atherosclerosis, especially among women. Since the genetic background of this F H population was diverse, it is possible that studies of other populations which are more homogenous may be more likely to reveal the effects of 49 the apo E polymorphism. It has been suggested that heterozygous FH should be referred to as a risk factor rather than a disease as a large fraction of those carrying the mutant gene, especially women, are not affected by symptoms of atherosclerosis (24). The results from the analysis of this FH population would also be consistent with such a viewpoint. From this study, it appears that the interaction of this "risk factor" with other environmental and genetic influences is more important when evaluating the risk of developing CAD. For example, a recent study of Lp(a) levels and apo(a) phenotype in 115 FH patients indicated that this polymorphism was a significant independant risk factor for the development of coronary heart disease (93). For this reason, further study of the co-inheritance of other genetic polymorphisms related to atherosclerotic risk should be pursued in order to better understand the clinical variation within FH. In this study, we have assessed the influence of selected genetic and environmental factors on the phenotypic expression of familial hypercholesterolemia. We have established that the dissimilarity in clinical expression between men and women is related to differences between the impact of known risk factors and the incidence of CAD. Even in the presence of a genetic mutation causing overt hypercholesterolemia, the expression of this disease is markedly affected by gender, lipid profile, smoking and hypertension. In our study, only a small number of women with FH had symptoms of coronary disease; however, the risk of developing CAD in females was significantly increased in the smaller fraction of patients who had hypertension or elevated triglycerides. In contrast, men had a higher general 50 frequency of disease but were even at a much greater risk if they had lower H D L cholesterol values and a history of smoking. 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